The long-term goal of this research initiative is to delineate the cytoprotective role of proinflammatory cytokines in myocardial ischemia reperfusion (I/R) injury. Studies from this laboratory have shown that the cytoprotective effects of tumor necrosis factor (TNF) are conveyed by both the type 1 (TNFR1) and type 2 (TNFR2) TNF receptors. Noting that the only signaling protein that was common to both receptors was a signaling protein termed TRAF2 (tumor necrosis factor receptor associated factor 2), we have performed experiments in isolated cardiac myocytes, as well as transgenic mice with cardiac restricted overexpression of TRAF2 (MHC-TRAF2 mice) and a dominant negative TRAF2 protein (MHC-TRAF2 mice) that have identified an indispensable role for TRAF2 in TNF mediated cytoprotective signaling. To further identify the mechanisms for the cytoprotective effects of TRAF2 we performed comparative gene expression profiling in the hearts of MHC-TRAF2 and MHC-TRAF2-DN mice and identified a unique "emergency response gene" termed dysferlin that is responsible for maintaining Ca++ dependent cardiac myocyte plasma membrane integrity. Given the central role of enhanced membrane permeability and Ca++ overload in the setting of ischemia reperfusion (I/R) injury, we propose to focus the present application on delineating the cytoprotective effects of TRAF2 signaling and will seek to address whether dysferlin is necessary and/or sufficient for mediating these cytoprotective effects. Accordingly, we propose to ask and answer a series of three logical questions. First, are the cytoprotective effects of TRAF2 mediated by activation of the type 1 NF-?B pathway, and if so are p65 and p50 homo or heterodimers involved (Specific Aim 1)? Second, is dysferlin necessary for the cytoprotective effects of TRAF2 following I/R injury (Specific Aim 2)? Third, what are the cellullar mechanisms responsible for cytoprotective effects of TRAF2 and/or dysferlin in isolated cardiac myocytes following hypoxia- reoxygenation injury (Specific Aim 3)? We expect that the results of Specific Aims 1 - 3 will provide definitive information with respect to the cytoprotective signaling pathways that are downstream from TRAF2 mediated in the heart, as well as allow us to determine whether dysferlin, a novel Ca++ dependent emergency response gene, is necessary and/or sufficient to mediate the cytoprotective effects of TRAF2. PUBLIC HEALTH RELEVANCE: Myocardial reperfusion following a period of ischemia may be regarded as a "mixed blessing." That is, on the one hand there is the clear cut benefit that occurs as the result of recovery of heart muscle that attends reperfusion;however, on the other hand there are also deleterious reperfusion dependent effects that limit the effectiveness of all reperfusion strategies, which can lead to heart failure. The significance of the studies proposed in the present application is that will (1) focus on identifying cytoprotective mechanisms that have been highly conserved in nature (signaling through tumor necrosis factor receptor associated factor 2 [TRAF2]), and (2) study a novel method for conferring cytoprotection in cardiac myocytes through enhanced membrane repair following ischemia reperfusion injury.